Characterization of Pathogenicity Genes in Colletotrichum scovillei Using Agrobacterium-Mediated Random Insertional Mutagenesis

Chili anthracnose, caused by Colletotrichum scovillei, is a destructive disease that significantly reduces both yield and quality in Capsicum annuum. Although highly virulent isolates such as CNU151001 have been identified, the molecular mechanisms underlying pathogenicity remain poorly understood. In this study, we employed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate insertional mutants of C. scovillei for the identification of virulence-associated genes. A total of 525 transformants were obtained, and pathogenicity assays on chili fruit identified 69 mutants with reduced virulence. Among these, mutants Csm265 and Csm414 consistently exhibited stable phenotypes across three rounds of TAIL-PCR analysis, which revealed T-DNA insertions in an uncharacterized gene predicted to encode a short transmembrane protein. Structural analysis indicated the presence of intrinsically disordered regions, suggesting potential roles in protein–protein interactions during host–pathogen communication. Additionally, phenotypic assays showed that mutant Csm265 produced significantly smaller conidia compared to the wild type, and GFP-tagging confirmed gene expression during spore development. These findings enhance our understanding of C. scovillei pathogenesis and provide novel candidate genes for future functional characterization of fungal virulence factors.